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A theory of topological constraints in polymer networks

Summary This paper presents an investigation of the dependence of harmonic-like topological constraints in entangled and moderately crosslinked polymer networks on segment and crosslink density and on the deformation of the sample. The approach is an extension of a theory developed for the highly crosslinked case and for polymer melts. The results are used to discuss the influence of topological constraints on the mechanical properties of different types of rubberelastic networks.     

OLN-93: a new permanent oligodendroglia cell line derived from primary rat brain glial cultures

We have established a new permanent cell line (OLN-93), derived from spontaneously transformed cells in primary rat brain glial cultures. In growth medium supplemented with 10% fetal calf serum a doubling time of 16-18 hr was determined. OLN-93 cells in their antigenic properties resemble primary oligodendrocytes in culture. As analyzed by indirect immunofluorescence, the A2B5 surface marker is absent, they express galactocerebroside and myelin-specific proteins, such as myelin basic protein (MBP), myelin-associated glycoprotein (MAG), proteolipidprotein (PLP), and Wolfgram protein (WP), but do not exhibit astrocytic properties, such as the expression of vimentin or the glial fibrillary acidic protein (GFAP). In their morphological features they resemble bipolar O-2A-progenitor cells and, when grown at low density or on poly-L-lysine-coated culture dishes under low serum conditions, immature oligodendrocytes with a more arborized cell morphology. The cellular processes contain microfilaments, while N-CAM/D2 immunoreactivity is localized on the cell surface of the somata and processes. Immunoblot analysis further confirmed the presence of MAG, WP and MBP immunoreactivity, and the absence of vimentin and GFAP. Only a single MBP isoform (approximately 14 kDa) was detectable in the cellular extracts. PLP mRNA expression was studied by RT-PCR. The two proteolipid-specific mRNAs, DM20 and PLP, were present in OLN-93 cell extracts. Comparisons with embryonic rat cerebral cells in culture and primary oligodendrocytes suggest that OLN-93 cells in their morphological features and their antigenic properties resemble 5- to 10-day-old (postnatal time) cultured rat brain oligodendrocytes. Thus, the new cell line described in this study should provide a useful model system to investigate the specific mechanisms regulating the proliferation and differentiation of oligodendrocytes in vitro, and the molecular interactions with other cells of the nervous system.     

Erfahrungen mit der Auswaschung von Kartoffelreibsel ohne Wasserzusatz

Experience in Potato Pulp Washing without Water Addition. In order to reduce the quantity of waste water from potato starch factories many have tried to keep the amount of water, required for washing out the starch from the pulp as low as possible. Our process is based on utilization of the water contained in the potatoes (approx. 80%, including soluble components) for starch extraction, in such a way that undiluted fruit water is separated and further treatment can be carried out in a comparatively economical way. Most of today's starch factories are tied up with production methods that make it impossible to utilize the water contained in the potatoes, as there is firstly no possibility of recirculating the fruit water and secondly there are foaming problems causing a negative effect on the efficiency. A continuous process guaranteeing a direct flow, excluding accumulation of foam, was installed and tested in both a Swedish and in a Dutch factory. The experience obtained showed that starch extraction with fruit water has no negative influence on the efficiency of the extraction (approx. 1 % free starch in the pulp DS/DS) and that up to 80% of the fruit water could be removed in a concentrated form. By extending the extraction process, and by using 300 l of process water from the refining section per ton of potatoes, separation of the fruit water can be increased up to 97%.     

The Functional Characterization of GCaMP3.0 Variants Specifically Targeted to Subcellular Domains

Calcium (Ca²⁺) ions play a pivotal role in physiology and cellular signaling. The intracellular Ca²⁺ concentration ([Ca²⁺]_i) is about three orders of magnitude lower than the extracellular concentration, resulting in a steep transmembrane concentration gradient. Thus, the spatial and the temporal dynamics of [Ca²⁺]_i are ideally suited to modulate Ca²⁺-mediated cellular responses to external signals. A variety of highly sophisticated methods have been developed to gain insight into cellular Ca²⁺ dynamics. In addition to electrophysiological measurements and the application of synthetic dyes that change their fluorescent properties upon interaction with Ca²⁺, the introduction and the ongoing development of genetically encoded Ca²⁺ indicators (GECI) opened a new era to study Ca²⁺-driven processes in living cells and organisms. Here, we have focused on one well-established GECI, i.e., GCaMP3.0. We have systematically modified the protein with sequence motifs, allowing localization of the sensor in the nucleus, in the mitochondrial matrix, at the mitochondrial outer membrane, and at the plasma membrane. The individual variants and a cytosolic version of GCaMP3.0 were overexpressed and purified from E. coli cells to study their biophysical properties in solution. All versions were examined to monitor Ca²⁺ signaling in stably transfected cell lines and in primary cortical neurons transduced with recombinant Adeno-associated viruses (rAAV). In this comparative study, we provide evidence for a robust approach to reliably trace Ca²⁺ signals at the (sub)-cellular level with pronounced temporal resolution.     

Smaller,Stronger, More Stable: Peptide Variants of a SARS-CoV-2 Neutralizing Miniprotein

Based on the structure of a de novo designed miniprotein (LCB1) in complex with the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, we have generated and characterized truncated peptide variants of LCB1, which present only two of the three LCB1 helices, and which fully retained the virus neutralizing potency against different SARS-CoV-2 variants of concern (VOC). This antiviral activity was even 10-fold stronger for a cyclic variant of the two-helix peptides, as compared to the full-length peptide. Furthermore, the proteolytic stability of the cyclic peptide was substantially improved, rendering it a better potential candidate for SARS-CoV-2 therapy. In a more mechanistic approach, the peptides also served as tools to dissect the role of individual mutations in the RBD for the susceptibility of the resulting virus variants to neutralization by the peptides. As the peptides reported here were generated through chemical synthesis, rather than recombinant protein expression, they are amenable to further chemical modification, including the incorporation of a wide range of non-proteinogenic amino acids, with the aim to further stabilize the peptides against proteolytic degradation, as well as to improve the strength, as well the breadth, of their virus neutralizing capacity.